Proliferation of smooth muscle cells in blood vessel walls occurs in response to vascular injury, and in association with certain disease states (Austin, G.E., et al., J Am Coll Cardiol (1985) 6:369-375). The proliferation of these cells can have negative effects due to the production of excess proteins or other matrix molecules, which, along with the cells themselves, form pathologic lesions of, for example, atherosclerosis, renal hypertension, pulmonary hypertension, vasculitis, and post-surgical vascular restenosis. These results are distinguished from the acute response to trauma characterized by blood clotting.
Heparin/heparan sulfate is known to inhibit smooth muscle cell proliferation. Heparin/heparan sulfate is a member of a class known as glycosaminoglycans (GAG). These materials are copolymers of alternating hexosamine and aldouronic acid residues which are found in sulfated forms and are synthesized as proteoglycans.
In the compositions of interest herein, heparan sulfate and heparin, the hexosamine is mostly N-acetylated or N-sulfated glucosamine (GlcNAc and GlcNS), and the aldouronic acid is mostly L-iduronic in heparin and mostly D-glucuronic acid in heparan sulfate. Heparan sulfate is commonly considered to have a higher proportion of glucuronic acid than heparin.
Problems of heterogeneity in preparations of heparan sulfate or heparin isolated from tissues make sharp distinctions difficult, since these oligosaccharides are related by their biosynthesis pathway, as explained below. Conventional heparin (used as an anticoagulant) has a molecular weight of 5-25 kd and is extracted as a mixture of various chain lengths by conventional procedures. These procedures involve autolysis and extraction of suitable tissues, such as beef or porcine lung, intestine, or liver, and removal of nonpolysaccharide components.
The molecular weight of the chains in the extract is significantly lower than the 60-100 kd known to exist in the polysaccharide chains of the heparin proteoglycan synthesized in the tissue. The GAG moiety is synthesized bound to a peptide matrix at a serine residue through a tetrasaccharide linkage region of the sequence D-GlcA-D-Gal-D-Gal-D-Xyl.fwdarw.protein, which is then elongated at the D-GlcA residue with alternate additions of GlcNAc and GlcA.
The polysaccharide side chains are modified by a series of enzymes which sequentially deacetylate the N-acetyl glucosamine and replace the acetyl group with sulfate, epimerize the hydroxyl at C5 of the D-glucuronic acid residue (to convert it to L-iduronic acid and the GAG chain from the heparan type to a heparin type), sulfate the O-2 of the resulting L-iduronic acid and the O-6 of the glucosamine residue. Some of the chains are further sulfated at the O-3 of the glucosamine residue, either at the heparan or heparin stage. This further sulfation is associated with the active site for binding to antithrombin III (ATIII) which is associated with anticoagulant activity. A synthetic pentasaccharide sequence capable of binding ATIII has been identified as ##STR2## by Choay (French Application No. 2,535,324). However it appears that the sequence in heparin corresponding to this pentasaccharide is generally ##STR3##
Other chemically possible sulfation sites are on the O-3 of L-iduronic or D-glucuronic and O-2 of D-glucuronic acid; however, these are seldom found.
Due to their obvious chemical similarity, isolated "heparin" may contain considerable amounts of what might otherwise be classified as heparan sulfate.
There is an extensive body of art concerning depolymerization of heparin/heparan sulfate chains and separation of products by size. In a generally used procedure, the heparin starting material is depolymerized in the presence of nitrous acid with or without pretreatment to remove acylation from any GlcNAc residues present. Nitrous acid, under the appropriate conditions, cleaves at the linkage between a GlcNS or GlcNH.sub.2 residue and the uronic acid residue through which it is linked through a glucosamine .alpha.(1-4) uronic acid linkage. If the heparin has been deacetylated, all of the glucosamine.fwdarw. uronic acid residues are susceptible and complete depolymerization results in disaccharides. If the heparin has not been deacetylated, the glucosamine.fwdarw. uronic acid residues wherein the glucosamine is acetylated are resistant, and both disaccharides and tetrasaccharides containing the resistant linkage result. In all cases, the glucosamine residue at the reducing terminus of the disaccharide or tetrasaccharide is converted to a 2,5-anhydromannose in the course of cleavage. This residue may further be reduced to the corresponding 2,5-anhydromannitol. These methods have been described by Bienkowski, M.J. and Conrad, H.E., J Biol Chem (1985) 260:356-365; Guo, Y., et al., Anal Biochem (1988) 168:54-62; and Guo, Y. and Conrad, H.E., Analyt Biochem (1989) 176:96-104. These methods are useful in analyzing the structure of heparin and in assessing the results of various treatments of the heparin chains. Further, there has been considerable attempt to use the products of degradation of heparin from both complete and partial digestion with nitrous acid as described in the foregoing papers, or from heparinase digestion or from periodate oxidation followed by .beta.-elimination. All of these processes generate low molecular weight heparins for therapeutic use.
The involvement of heparin or heparan sulfate or degradation products thereof in smooth muscle proliferation has been recognized for some time. Heparin and heparan sulfate can slow or arrest the vascular proliferation associated with injury described hereinabove (Clowes, A.W., et al., Nature (1977) 265:625-626). The effect of heparan sulfate and heparin on smooth muscle proliferation is also described by Marcum, J.A., et al. in Biology of Proteoglycan, Academic Press (1987) pp. 301-343 The inhibition of vascular smooth muscle cell growth by heparin was further described by Castellot, J.J., Jr., et al., J Biol Chem (1982) 257:11256-11260, and the effect of heparin on vascular smooth muscle cell growth in fetal tissue was described by Benitz, W.E., et al., J Cell Physiol (1986) 127:1-7. The effect of heparin as an inhibitor of both pericyte and smooth muscle cell proliferation was shown by Orlidge, A., et al., Microvascular Research (1986) 31:41-53, and these authors further showed that chondroitin sulfate, and dermatan sulfate do not have this effect. A review of the effects of heparin and heparan sulfate on the proliferation of smooth muscle cells has been published by Benitz, W.E. in "The Pulmonary Circulation: Normal and Abnormal", Fishman, A.P., ed., University of Pennsylvania Press (1988).
It is not clear by what mechanism these glycosaminoglycans operate, or to what extent they interact with other growth factors such as epithelial and fibroblast growth factors. It has been proposed that a 3-O sulfate on glucosamine in an oligosaccharide of at least 5 sugars is important in this process and that both O-and N-sulfation is important (Castellot, J.J., et al., J Cell Physiol (1984) 120:315-320; Castellot, J.J., et al., J Cell Biol (1986) 102:1979-1984). Hexasaccharides-decasaccharides obtained from partial nitrous acid digestion of heparin bind to acidic fibroblast growth factor and aid its mitogenic activity in fibroblasts, but inhibit the proliferation of endothelial cells under some conditions (Barzu, T., et al., J Cell Physiol (1989) 140:538-548). The effective hexasaccharide was stated to have the structure: ##STR4##
Others have indicated that the presence of 2-0-sulfate glucuronic acid is not necessary for antiproliferative activity (Wright, Jr., T.C., et al., J Biol Chem (1989) 264:1534-1542). In this article, size separated fragments of defined length prepared by nitrous acid cleavage and gel filtration were further separated according to charge for some assays. Partially digested heparin separated only according to size was tested with respect to stimulation of smooth muscle cells and epithelial cells. Similar results were found in both cases, although the results were not identical. Tetrasaccharides of the type tested were shown to have very low antiproliferative activity; hexasaccharides, octasaccharides and decasaccharides were shown to be active to approximately the same level on a weight/volume concentration basis. Also tested was a synthetic pentasaccharide which represents a unique sequence of the heparin-binding site in heparin to antithrombin III; this pentasaccharide was active in inhibiting proliferation for smooth muscle cells, but not for epithelial cells.
The size separated fractions were then treated chemically to produce "O-oversulfation" and this treatment enhanced the inhibitory activity; indeed, O-oversulfation of the tetrasaccharide fragment preparation caused the tetrasaccharide fraction to become active in inhibiting proliferation. The converse process, comprising desulfation and reacetylation of the amino groups or glucosamine results in a reduction in antiproliferative activity. These fragments could, however, be made more active by subsequent O-oversulfation.
Also capable of reducing the antiproliferative activity of the heparin fragments was reduction of the carboxyl groups so as to reduce the total negative charge. O-oversulfation partially, at least, restores this activity. These results with N-desulfated, N-acetylated fragments which are lacking in antiproliferative activity are distinguishable from previous results wherein similarly treated heparin retains the capacity to prevent cell division because of the size dependency of the antiproliferative activity-larger fragments being more powerful in general than smaller ones.
Finally, when the size separated fraction was then further fractionated according to charge, it was found that the most highly charged fractions showed the greatest activity. Furthermore, it was shown that although the synthetic pentasaccharide identified with the antithrombin III binding site is capable of inhibiting proliferation in smooth muscle cells, treatment of heparin which would destroy the sequence corresponding to this pentasaccharide (i.e., periodate treatment) does not destroy antiproliferative activity. As stated above, this synthetic pentasaccharide has the structure: ##STR5##
U.S. Pat. No. 4,990,502 describes the treatment of heparin with periodate, followed by depolymerization with base, and reduction of the aldehydes generated in the periodate treatment. The resulting material is said to contain a mixture of polymers containing 17-33 residues and containing a multiplicity of residues of the formula ##STR6## wherein the glucosamine residue is sulfated at the 2 and/or 6 position in an arbitrary manner, and wherein some of the IdoA residues may be replaced by cleaved IdoA ##STR7## or GlcA residues resulting from the periodate oxidation. These shortened polymeric chains are said to lack the binding site for ATIII but to be capable of inhibiting smooth muscle proliferation and to have physiological activities that include acceleration of tissue repair, prevention of atherogenous lesions, prevention of states of shock, and prevention of the development of metastasis.
Treatment of heparin/heparan sulfate with periodate has also been reported by others. Fransson, L.-A. and Lewis, W., FEBS Lett (1979) 97:119-123, describe a variety of conditions relating to the treatment of heparin/heparan sulfate with periodate and reduction by sodium borohydride or fragmentation in alkaline medium. Fransson concluded (erroneously as will be shown hereinbelow) that the glucuronic acid residues were preferentially oxidized as compared to idouronic acid residues, and that complete cleavage of all susceptible uronic acid residues, which is said to result in pronounced fragmentation of the molecule, resulted in the absence of anticoagulant activity. Fransson, L.-A. et al., Carbohydrate Res (1980) 80:131-145, studied chemistry of various forms of treatment of heparin with periodate. In one study, the treatment with periodate is followed by .beta.-elimination in base to produce fragmentation. They further report treatment of heparin with periodate followed by partial acid hydrolysis which results in fragmentation of the chains and partial destruction of the functional groups.
Casu, B. et al., Arzneim Forsch/Drug Res (1986) 36:637-642, studied the effect of periodate oxidation on the anti-lipemic (lipoprotein lipase-releasing) activity of heparin. In this study, the heparin was oxidized with periodate and the products were reduced with borohydride without depolymerization. The resultant was said to have the same molecular weight as the starting material. Although the ATIII binding activity of the treated material was greatly diminished, the anti-lipemic activity was said to be maintained. The amount of reduction in anticoagulant activity was said to be less for heparin derived from beef lung than that derived from porcine mucosa.
In addition to activities in releasing lipoprotein lipase and in inhibiting smooth muscle cell proliferation, heparin has been shown to inhibit platelet aggregation. This has been evidenced by the ability of heparin to prolong the bleeding time in animals. Indeed, the interference with platelet aggregation is thought to lead to an undesirable side effect of anticoagulant treatment with heparin, namely a bleeding liability with respect to some patients.
It will be noted that heparin is a complex molecule with a complex array of activities in vivo. While a particular subunit, specifically a pentasaccharide, has tentatively been designated as responsible for anticoagulant activity, heparin is also known to bind to a variety of growth factors to mediate or inhibit growth of various cell types, and may provide additional functions as yet to be ascertained. The overall structure of the molecule may be important in some degree in some or all of these. Also, the polymers generally are expected to have multiple binding sites which results in a bonding affinity not generated by a smaller fragment. Thus there is advantage in maintaining the integrity of the heparin molecule to the extent possible when destroying undesirable functions, i.e., anticoagulation properties.
The present invention provides inactivation of the anticoagulant ability of heparin without destruction of antiproliferative activity without fragmentation of the heparin chains, thus preserving to the extent possible desirable additional functions. This process has the additional advantage of retaining the size distribution of the naturally-occurring heparin/heparan sulfate preparation, which results in a therapeutic having a more readily recognized physiological profile.